The determination of the molecular weight of molecules within a sample may be an important first step either in determining the presence of a known molecule in a sample or in determining the structure of an analyte of unknown structure. Various techniques may be employed to determine the molecular weights of analytes depending upon the degree of precision required and the characteristics of the analyte itself. Thus, electrophoresis, centrifugal sedimentation, and mass spectrometry may all find use in different circumstances. Whereas electrophoresis sedimentation provide some measure of accuracy in estimates of molecular weight, mass spectrometry provides for much greater accuracy.
Soft ionization mass spectrometry techniques include fast-atom or ion bombardment (FAB) ionization spectrometry, electrospray spectrometry, plasma desorption mass spectrometry (PDMS), and matrix-assisted laser desorption ionization (MALDI) spectrometry. MALDI, for example, permits the determination of the molecular weight of proteins up to the 10.sup.5 Da range with an accuracy of 0.1-0.01%, requiring only picomoles or sub-picomoles of material (1-4). The method is equally applicable to smaller biologically important molecules such as peptides (5), carbohydrates (6), oligonucleotides (7,8), glycolipids (9), and polar and nonpolar synthetic polymers (10,11). It has become an important technique in biochemistry and biology not only because the molecular weight of the native material at that level of accuracy is in itself very useful information, but also because the changes thereof upon chemical or enzymatic treatment provide further insight into the structure or biological significance of parts of the native molecule (12). These manipulations are often necessary to obtain structural information because limited excess energy is transferred to the analyte during the MALDI process and "prompt" fragmentation is therefore rarely observed. This feature is an advantage in the analyses of mixtures, as long as the components can be resolved.
Although most of the compounds in the above-mentioned categories are amenable to mass spectrometry, several difficulties arise when the analyte is highly polyionic (i.e. highly polyacidic or highly polybasic). In the first instance, it may simply be difficult to ionize such analytes. Highly acidic compounds, for example, are difficult to ionize even in the negative mode of a mass spectrometer where they are detected as anions. Although attempts have been made to analyze highly polyacidic compounds in the negative mode, most of these efforts have been devoted to oligonucleotides (7,8). It is even more difficult to ionize polysulfate esters or polysulfonic acids. This is due, in part, to the fact that these substances tenaciously attach cations (such as Na.sup.+, K.sup.+, etc.) to form a multiplicity of analyte-cation complexes. These complexes give rise to broad unresolved peaks in mass spectra, the centroid of which corresponds to the average mass of all these partial salts.
Peptidoglycans (PG) and glycosaminoglycans (GAG) are examples of polyacidic molecules of great biological significance that have been difficult to analyze. Despite their abundance in living organisms as constituents of the extracellular matrix or cell surfaces, and their extensive use in medicine (most importantly, heparin), even the primary structures of some of these highly polar and polydisperse compounds are not well-understood (19,26). In addition to their tendency to form complexes with small cations, these compounds are characterized by variable degrees of sulfation. This is characteristic of, for example, glycosaminoglycans composed of uronic acid and glucosamine residues: heparin, heparan sulfate, dermatan sulfate and chondroitin sulfate. As a result, in contrast to the level of detail with which gene sequences can be determined, even the primary sequences for the GAGs heparin and heparan sulfate are not known. To date, only typical and/or abundant subsequences of GAGs have been characterized by affinity and sizing chromatography of GAG degradation products (27-34).
Mass spectrometry is a particularly useful and general analytical method for problems where structural regularities of the material being investigated allow one to deduce structural details from molecular weight information. This is certainly the case with the GAGs heparin and heparan sulfate, where accurate mass measurement (with, for example, .+-.0.05% uncertainty) unambiguously identifies oligosaccharides except for structural isomers. Some of these isomeric ambiguities may then be resolved by specific enzymatic reactions. Presumably due to the difficulties of ionizing these compounds in a mass spectrometer, few mass spectrometric studies of GAGs have been reported. Plasma desorption mass spectrometric (PDMS) studies were carried out by McNeal et al. (35), where data on the molecular weights and extent of sulfation were determined for heparin-derived oligosaccharides up to hexasaccharides from 25-50 .mu.g samples (20-30 nmol). Ten nmol sensitivity was reported by Carr and Reinhold (36,20) for chondroitin sulfate oligosaccharides and synthetic heparin oligosaccharides up to pentamers studied by fast atom bombardment (FAB) ionization in the negative ion mode. Somewhat improved performance was obtained by Mallis et al. (21,22) who were able to detect heparin-derived oligosaccharides up to octamers using triethanolamine as FAB matrix rather than the thioglycerol employed earlier by Carr et al. (36). More recently, electrospray studies were conducted on disaccharides with further improved sensitivity (100 pmol level) (37). All of these efforts are characterized by low sensitivity (compared to that of peptides and proteins), by abundant multiple adducts of alkali cations and by partial elimination of the sulfate groups. These features interfere with the unambiguous identification of individual components and with the analysis of heterogeneous mixtures at high sensitivity.
In one attempt to improve the accuracy of mass spectrometric mass determination of heparin fragments, an immobilized cationic surfactant was used to displace, in part, sodium cations from complexes with the analyte (35). The surfactant, triddecylmethyl ammonium chloride (TDMAC), formed complexes with the analyte which somewhat increased sensitivity and resolution. TDMAC, however, is a fixed-charge monobasic ion and, as such, forms a multiplicity of complexes with polyionic analytes in which some labile groups are unprotected by ionic bonding. Thus, fragmentation was observed, meaningful mass estimates were difficult to determine, and samples of analyte in the 25-50 .mu.g range were needed.